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Guidelines for BAM Users on Identification of Atypical Hemolytic Listeria Isolates

It is recommended that the identities of all atypical isolates of hemolytic Listeria, which do not produce acid from L-rhamnose and D-xylose in single carbohydrate tests, be confirmed either as L. monocytogenes or as not-L. monocytogenes by additional testing. The reason for this recommendation is that at least one naturally occurring hemolytic Listeria strain is documented, which does not segue easily into the current taxonomic scheme by conventional testing (4). Selected taxonomically relevant common and atypical phenotypes of Listeria are listed in Table 1. Included is the phenotype of the difficult to speciate strain, which is partial hybrid of L. monocytogenes with another species, L. innocua (7). Although the chance of isolating a strain like this one is relatively low, its incorrect species assignment could be an issue.

Table 1. Taxonomically Relevant Listeria Phenotypesa
Species Phenotype Comments & references
L. monocytogenes Hly+, Rha+, Xyl- Normal biotype (2,3,5)
Hly+, Rha-, Xyl- Uncommon biotype (6,9)
Hly-, Rha+, Xyl- Uncommon biotype (1,5)
L. innocua Hly-, Rha+, Xyl- Common biotype (2,3,5)
Hly-, Rha-, Xyl- Common biotype (2,3,5)
Hly+, Rha-, Xyl- Hybrid-like strain (4,7)
Hly+, Rha+, Xyl- No reported example
L. innocua-like Hly+, Rha-, Xyl- Nominally hemolytic L. innocua strains (3) b
L. seeligeri Hly+, Rha-, Xyl+ Normal biotype (2,3,5)
Hly+, Rha-, Xyl- Uncommon biotype (8)
Hly-, Rha-, Xyl+ Unpublished data

a Phenotype abbreviations: Hly = hemolysis; Rha = Acid from L-rhamnose; Xyl = Acid from D-xylose. Bold abbreviations indicate the uncommon biotype.
b Pragmatically, FSIS (3) nominally categorizes an isolate with the Hly+, Rha-, Xyl- phenotype as a hemolytic L. innocua strain if the strain is negative for L. monocytogenes-specific r-RNA by the AccuProbe or Gene-Trak tests. An infrequent biotype of L. seeligeri (8) has the same phenotype but further taxonomic resolution is unnecessary for the purpose of ruling out the Hly+, Rha-, Xyl- phenotype of L. monocytogenes (L. V. Cook, personal communication).

The easiest way to confirm the identities of aberrant isolates is to see whether the isolate is DIM negative (L. monocytogenes) or DIM positive (the other Listeria species). The DIM [Differentiation /Innocua /Monocytogenes] colorimetric test for naphthylamidase activity is a component of the API (bioMerieux, Inc) test module for identifying Listeria species. The DIM result may already be known in the course of conventional speciation since use of the kit is a prescribed choice in the BAM Listeria monocytogenes analysis. Although no DIM positive varieties of L. monocytogenes have been reported, DIM negative strains (8) of the characteristically DIM positive species do exist. Thus a DIM-positive result shows the isolate is not L. monocytogenes but a DIM-negative result only means a putative L. monocytogenes identity.

The preferred confirmation method, which gives a positive test result for L. monocytogenes, is the use of a DNA probe test for L. monocytogenes specific r-RNA, which is a highly conserved target. No confirmable false negatives have been reported to date. Commercial kits are available for this test (Gene-Trak Inc. or GenProbe Inc.).

Contact Karen.Jinneman@fda.hhs.gov for assistance with DNA-probe testing of the r-RNA of any unusual Listeria isolates and about contributing such isolates to the collection of aberrant strains at the Listeria Methods Research Laboratory.


  1. ATCC. 1987. Listeria monocytogenes. Catalogue of Bacteria & Bacteriophages. 17th Ed. p. 124. American Type Culture Collection, Manassas VA.
  2. BAM Chapter 10: Detection and Enumeration of Listeria monocytogenes (accessed 04/15/2011).
  3. FSIS method for the isolation and identification of Listeria monocytogenes from processed meat and poultry products (PDF, 2.0 Mb): Chapter 8.07 from the USDA FSIS Microbiology Laboratory Guidebook (accessed 04/15/2011).
  4. Johnson, J., K. Jinneman, G. Stelma, B.G. Smith, D. Lye, J. Messer, J. Ulaszek, L. Evsen, S. Gendel, R. W. Bennett and A. D. Hitchins. 2000. Atypical Hemolytic Strain of Listeria Difficult to Speciate by a Battery of Accepted Methods. Abstract. AOAC Intl. Ann. Mtg., Philadelphia, PA. p. 24.
  5. Rocourt, J. 1999.The genus Listeria and Listeria monocytogenes: phylogenetic position, taxonomy, and identification. Pp. 1-20. In E. T. Ryser and E. H. Marth (eds) Listeria, Listeriosis, and Food Safety, Marcel Dekker, Inc., New York and Basel.
  6. Siragusa, G. R., J. S. Dickson, and E. K. Daniels. 1993. Isolation of Listeria spp. from feces of feedlot cattle. J. Food Protect. 56:102-105, 109.
  7. Johnson, J., Jinneman, K., Stelma, G., Smith, B.G., Lye, D., Messer, J., Ulaszek, J., Evsen, L., Gendel, S., Bennett, R. W., Swaminathan, B., Pruckler, J., Steigerwalt, A., Kathariou, S., Yildirim, S., Duvall, R. E., and Hitchins, A. D. 2003. Discovery of a Hemolytic Listeria innocua Strain. FDA Science Forum Abstracts. Page 163, No. P-PO-1.
  8. The api Listeria system for the identification of Listeria. Numerical profiles list. Kit package insert. bioMérieux SA Paris, France.
  9. Wiedmann, M., Bruce, J. L., Keating, C., Johnson, A. E., McDonough, P. L., and Batt, C. A. 1997. Ribotypes and virulence gene polymorphisms suggest three distinct Listeria monocytogenes lineages with differences in pathogenic potential. Infect. & Immun. 65:2707-2716. 

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